1. Field of the Invention
The invention relates to the field of biological reactions which are carried out at two or more different temperatures. More particularly, it relates to chain reactions for amplifying DNA or RNA (nucleic acids), or other nucleic acid amplification reactions, e.g., Ligase Chain Reaction (LCR), or reverse transcription reactions and methods for automatically performing this process through temperature cycling. This invention also relates to thermal cyclers for automatically performing this process through temperature cycling
2. Description of the Prior Art
Thermal cyclers may be used to perform Polymerase Chain Reaction (PCR), methods or other nucleic acid amplification reactions, e.g., Ligase Chain Reaction (LCR). Typically, there are three temperature-dependent stages that constitute a single cycle of PCR: template denaturation (95° C.); primer annealing (55 C 65° C.); and primer extension (72° C.). These temperatures may be cycled for 40 times to obtain amplification of the DNA target.
Some thermal cycler designs vary the temperature of a heat source to achieve denaturation, annealing, and extension temperatures. For example, U.S. Pat. No. 5,656,493 issued Aug. 12, 1997 to the Perkin-Elmer Corporation describes a heating and cooling system that raises and lowers the temperature of a heat exchanger at appropriate times in the process of nucleic acid amplification. A reaction vessel is embedded in the heat exchanger, and heat is transferred to the reaction vessel by contact with the heat exchanger. The disadvantage of such a system is that it takes time to raise and then to lower the temperature of the heat exchanger. This lengthens the time required to perform PCR.
Other designs use fixed-temperature heat blocks, and move the reaction vessel in and out of contact with the appropriate heat blocks. By saving the time required to ramp the temperature of the heat blocks, reactions may be performed in shorter times. For example, U.S. Pat. No. 5,779,981 issued Jul. 14, 1998 to Stratagene describes a thermal cycler which uses a robotic arm to move reaction vessels into contact with heat blocks set at fixed denaturation, annealing, and extension temperatures. For example, PCR may be performed with heat blocks set at fixed temperatures of 95° C., 55° C., and 72° C., respectively. The disadvantage of this system is that a separate heat block is required for each temperature setting. Each heat block takes up space and requires its own electrical control. As well, some applications may require more temperature settings than there are heat blocks. For example, the AgPath-ID™ One-Step RT-PCR Kit (Ambion) performs reverse transcription at 45° C. After reverse transcription, the reaction components may be used immediately for a 3-temperature PCR. However, if there are only three fixed-temperature heat blocks, then it will take time for one of the blocks to ramp from 45° C. to one of the three temperatures for PCR.
To minimize evaporative loss and undesirable condensation, the reagents in the reaction vessel may be overlaid with mineral oil. Alternatively, U.S. Pat. No. 5,552,580 issued Sep. 3, 1996 to Beckman Instruments Inc discloses the use of a heated lid to minimize condensation in instruments for DNA reactions.
The invention in its general form will first be described, and then its implementation in terms of specific embodiments will be detailed with reference to the drawings following hereafter. These embodiments are intended to demonstrate the principles of the invention, and the manner of its implementation. The invention in its broadest sense and more specific forms will then be further described, and defined, in each of the individual claims which conclude this Specification